Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
  • A21D8/00—Methods for preparing or baking dough
  • A21D8/02—Methods for preparing dough; Treating dough prior to baking
  • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
  • A21D8/045—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with a leaven or a composition containing acidifying bacteria
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
  • A21D8/00—Methods for preparing or baking dough
  • A21D8/02—Methods for preparing dough; Treating dough prior to baking
  • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
  • A21D8/047—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/819—Fermentation vessels in series
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/853—Lactobacillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/853—Lactobacillus
  • Y10S435/855—Lactobacillus brevis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/853—Lactobacillus
  • Y10S435/857—Lactobacillus plantarum
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi
  • Y10S435/94—Saccharomyces
  • Y10S435/942—Saccharomyces cerevisiae

Definitions

• This invention refers to a process for preparing a biomass consisting of yeast and lactic acid bacteria, to the use of the biomass so prepared as panification ferment, as well as to a panification ferment.
• a first object of the present invention is to eliminate said disadvantages of the prior art.
• Another object of the present invention is to create a biomass consisting of yeast and lactic acid bacteria, based on a cereal medium, the obtained biomass being ready to be used as panification ferment without preliminary separation of the culture medium and the biomass.
• the process according to the present invention permits to obtain defined mixtures of identified microorganisms, selected for their metabolical particularities, especially in the metabolism of sugars.
• the obtained products are well defined mixtures, allowing--depending on the nature of the selected strains and their representativeness in the mixture--to obtain, in the direct panification of the industrial type, products which are comparable to those obtained with sponges of the traditional type, and this in a perfectly reproducible manner. These mixtures can also be used in the home bakery with the same advantages.
• the culture is preferably of the discontinuous fated type, so called "fed-batch".
• This technique consists in continuously adding, in repeated periods, the necessary ingredients to a batch of the medium.
• This type of fermentation allows to control the metabolism of the culture between the aerobic and the anaerobic pathway, thus controlling the metabolic flux between growth (biomass) and the products derived from its metabolism (especially ethanol).
• the ethanol content of the culture can be controlled by regulating the alimentation speed to the culture medium, and the partial pressure of the oxygen dissolved in the culture can be controlled by regulating the air supply in accordance with a previously defined slope, maintaining the culture in a metabolism limited in oxygen.
• the culture can develop without pH regulation, thus avoiding the addition of any chemical additives.
• the coculture is grown as a mixed sequential culture. This means that microorganisms are added deferred in time, permitting to reutilize, at a given time, the products of the metabolism of the microorganisms previously present.
• the culture process is controlled so as to regulate the ethanol content between 0.5 and 10 grams/liter of culture.
• the process according to the present invention is executed as follows:
• a first culture medium (called “base medium”) is introduced into a bioreactor;
• this first culture medium is inoculated with the yeast and the lactic acid bacteria to be cultivated, distributed in a second culture medium (called “dosage medium”);
• the bioreactor is continuously fed with the second culture medium
• the obtained product can be used immediately after it left the bioreactor as ferment in the direct industrial panification without any further action, i.e. without previous separation of the culture medium and the biomass.
• the used yeast is a strain of Saccharomyces cerevisiae, preferably a strain isolated from a natural sponge, and particularly the strain Saccharomyces cerevisiae steineri DSM 9211. This strain was isolated from a home made sponge of excellent organoleptic quality. The characteristics of the type Saccharomyces cerevisiae are compiled in Table 1.
• Saccharomyces cerevisiae steineri strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9211.
• the lactic acid bacteria used are of the type Lactobacillus, Leuconostoc and/or Pediococcus, preferably a strain of Lactobacillus brevis, Lactobacillus plantarum, Leuconostoc mesenteroides and/or Pediococcus pentosaceus, and in particular one or several of the strains Lactobacillus brevis DSM 9209, Lactobacillus plantarum DSM 9208, Leuconostoc mesenteroides DSM 9207 and Pediococcus pentosaceus DSM 9210. These strains were isolated from the same sponge which was the base for the isolation of the above-mentioned strain Saccharomyces cerevisiae steineri. The characteristics of these strains are compiled in Table 2.
• the Leuconostoc mesenteroides strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9207.
• Taxonomy of Leuconostoc mesenteroides DSM 9207
• Lactobacillus plantarum strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9208.
• Lactobacillus brevis strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9209.
• the Pediococcus pentosaceus strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9210.
• Taxonomy of Pediococcus pentosaceus DSM 9210
• the strain of yeast is grown in a bioreactor in a mixed and/or sequential system in a discontinued fated culture with one or several lactic acid bacterial strains, depending on the nature of the desired panification product.
• the process according to the present invention permits to individually control the metabolism of each microorganism, allowing to act on the concentration of the final metabolites, particularly of the ethanol, the lactic acid and the acetic acid.
• the choice of the microorganisms on the basis of their metabolic particularities, particularly the homofermentative or heterofermentative metabolic pathway, and notably the regulation of the depending enzymatic activity of pyruvate oxydase oxygen (cf.: Frey, Le Lait 1992) allows to control with great precision the concentration of the acetic acid in the final mixture.
• the process according to the present invention allows to defer in time (sequential system) the bringing into action of one microorganism with respect to another.
• the growth of the lactic acid bacteria can start simultaneously (several strains inoculated at the same time) or in a differed way (a first strain put into coculture with the yeast or yeast, respectively, at time t 1 , then a second strain is introduced at time t 2 . . . t x ).
• the culture of the lactic acid bacteria can precede the start of the coculture with the yeast or several yeasts.
• Table 3 summarizes these possibilities in a simplified schema.
• L.a.b. Lactic acid bacteria
• Yeast autolysate of food quality providing the medium with vitamins and amino acids.
• a typical analysis of the product is as follows:
• the mixture is heated to 85° C. for 20 minutes and then cooled to 75° C. Thereafter, 2 milliliters of the same enzyme are added. The temperature is maintained for 20 minutes.
• the mixture is cooled to 60° C. Then, 50 milliliters of a solution of amyloglucosidase (16,700 AGI/milliliter) are added. The action of the enzyme is maintained for 90 minutes.
• the mixture is cooled to 50° C. and subjected to the hydrolysis by two specific proteases, the first one being purified and fractionated papaine, and the second one being fractionated pancreatine.
• the obtained culture medium is sterilized at 120° C. for 20 minutes.
• This medium which is perfectly stable is stored at +4° C.
• This medium serves for the inoculation and is called in technical terms "tank bottom” in the production system of the biomass according to the process of the discontinuedatoryd culture ("fed-batch").
• the medium is subjected to hydrolysis by alpha-amylase (2 milliliters/kilogram of wheat germs) for 20 minutes at 75° C., thereafter to the action of the above-mentioned two specific proteases, i.e. purified and fractionated papaine and fractionated pancreatine, at 50° C. for 240 minutes.
• the medium is conserved at a +4° C.
• alpha-amylase in two lots to the medium called "dosage medium” avoids the irreversible gelatinisation of the starch at the moment when the temperature raises above 65° C.
• the liberated sugars are measured by high performance liquid chromatography, and the amino acids liberated by the hydrolysis under the action of the protease are measured by means of the ninhydrine reagent (S. Moore and W. H. Stein, J. Biol. Chem. 176, 367, 1948).
• the final pH of the medium is near to 6.0, for example 5.5 to 6.5.
• This example describes a culture of the type (1) in Table 3.
• microorganisms are kept under conventional conditions in the laboratory and transplanted twice onto the previously defined medium, in order to adapt these microorganisms to the culture medium.
• the yeast is kept at -80° C. in a cereal medium containing glycerol. It is reisolated on a cereal medium. An isolated colony is put to growth in a liquid cereal medium (dosage medium diluted with one half of water).
• a second culture is made from the first one in an Erlenmeyer of 200 milliliters.
• the cellular density after 16 hours of culture at 30° C. with agitation is 3 ⁇ 10 8 cells/milliliter.
• a third culture is made from the second one in 600 milliliters inoculated by 20 milliliters of the previous culture.
• the cellular density obtained after 8 hours of culture at 30° C. is 2.5 ⁇ 10 8 cells/milliliter.
• This culture is used to inoculate a 15 liter bioreactor, having an useful capacity 10 liters.
• the measured ethanol content is 25 grams/liter.
• the glucose is completely metabolized.
• strains selected for their metabolism and their organoleptic characteristics, are preserved at -80° C. At the time of use, these strains are transplanted onto the solid cereal medium.
• the first culture is made from a isolated colony put into 100 milliliters of medium.
• the cellular density attained after 24 hours of culture at 30° C. is 1 to 5 ⁇ 10 9 cells/milliliter, depending on the used type.
• the second culture is made in a volume of 900 ml of a new medium inoculated with 100 milliliters of the previous culture. This culture is used for inoculating the bioreactor at the desired time and at the desired concentration.
• the ultimate aim which conditions the start of the process according to the present invention, is to obtain a preparation composed by a mixed culture allowing the preparation of a bread comparable to a bread made with a traditional sponge.
• the mixed microbial growth brings about numerous interactions of the type commensalism, mutualism and ammensalism between the strains involved.
• the process described hereafter tries to make a compromise in producing Lactobacillus in sufficient quantity to produce flavors and a characteristic acidity to the breads, and to secure a sufficient proportion of yeasts (rising agents) which guarantee a well aerated bread with good density without further addition of yeast.
• the culture starts with the growth of yeast. 5 liters of base medium and 600 milliliters of a yeast culture in an Erlenmeyer are continuously introduced into a 15 liter bioreactor, having a useful capacity of 10 liter. The mixture is fed with the dosage medium, and the temperature is maintained at 30° C. The pH, which is continuously measured, is not regulated. At the start the pH is 6, and it goes to 4.0 to 5.0 at the end of the mixed culture, depending on the strain of lactic acid bacteria used. The ethanol content is maintained between 0.5 and 10 grams/liter, by submission of the alimentation of the dosage medium.
• the agitation is effected by means of two agitator blades of the Rushton type (Size of the blades: one third of the diameter of the bioreactor--Transfer coefficient: 600 mmoles O 2 /liter--Agitation speed: 500 to 1,200 rpm--Exit of sterile air: varying from 0 to 30 liters/minute). Said air exit depends on the partial pressure of oxygen which is continuously measured by means of a pO 2 electrode (Ingold), and is kept above 10%.
• the delayed inoculation of lactic acid bacteria allows to adjust the concentrations of various microorganisms and to influence thereupon the organoleptic qualities of the final product.
• Table 8 shows a sequential culture in which Lactobacillus plantarum is added to the yeast culture 8 hours after its inoculation.
• This example shows a coculture which consists, at the start, in a mixed culture of yeast and Leuconostoc mesenteroides. This mixed culture is followed for 8 hours. After these 8 hours a pre-culture of Lactobacillus plantarum is added. The final product is obtained after 18 hours.
• This example shows a coculture of the same type as that of Example 2, but in this test Leuconostoc mesenteroides is replaced by Lactobacillus brevis in the coculture at the start. Lactobacillus plantarum is added after 8 hours.
• the obtained cellular density ratios between yeasts and bacteria are 3 to 5 (10 9 yeasts to 3 to 5 ⁇ 10 9 bacteria).
• the final culture is cooled to 3° C. as quickly as possible. It can be stored for 8 days without loosing its panification capacity. It is used in a concentration of 20% (weight/volume).

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• 1994
  • 1994-05-27 AT AT94810306T patent/ATE187770T1/de not_active IP Right Cessation
  • 1994-05-27 DK DK94810306T patent/DK0684308T4/da active
  • 1994-05-27 DE DE69422161T patent/DE69422161T3/de not_active Expired - Lifetime
  • 1994-05-27 EP EP94810306A patent/EP0684308B2/fr not_active Expired - Lifetime
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• 1995
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• 1997
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